Dna methylation detection methods

ABSTRACT

The present teachings provide DNA methylation quantification methods that avoid bisulfite treatment of DNA. Methylation-specific binding proteins (MeDNA binding proteins) and non-methylation specific binding proteins (non-MeDNA binding proteins) are employed in various embodiments to modulate the accessibility of nucleic acids to primer extension reactions. After selectively removing the target nucleic acids, the extension products can be analyzed and methylation quantitated. In some embodiments, the analysis comprises real-time PCR.

FIELD

The present teachings pertain to methods and kits for quantitatingcytosine methylation in target nucleic acids.

INTRODUCTION

Epigenomic changes to DNA provide another channel of information onwhich natural selection can act (see Goldberg et al., Cell, 128:635-638). Increasing attention is being paid to methylation of bases innucleic acids as one important epigenomic change. Methylation of basescan take different forms. For example, methylation of DNA by the DNAadenine methyltransferase (Dam) provides an epigenetic signal thatinfluences and regulates numerous physiological processes in thebacterial cell including chromosome replication, mismatch repair,transposition, and transcription (see Heusipp et al., Int J MedMicrobiol. 2007 February; 297(1):1-7. Epub 2006 Nov. 27 for a review).Also, methylation of cytosine in mammals at CpG dinucleotides correlateswith transcriptional repression, and plays a crucial role in generegulation and chromatin organization during embryogenesis andgametogenesis (Goll and Bestor (2006) Annu. Rev. Biochem. 74, 481-514).

One method of measuring the presence of cytosine methylation takesadvantage of the ability of the converting agent bisulfite to convertnon-methylated cytosines to uracil (See Boyd et al., Anal Biochem. 2004Mar. 15; 326(2):278-80, Anal Biochem. 2006 Jul. 15; 354(2):266-73. Epub2006 May 6, and Nucleosides Nucleotides Nucleic Acids. 2007;26(6-7):629-34. After such conversion, a sequence amplified in a PCRbears thymine at those residues that were originally unmethylatedcytosine. However, methylated cytosines are protected from suchbisulfite treatment. Accordingly, the presence of a thymine at alocation known to normally contain cytosine reflects that the originalcytosine was unmethylated. Conversely, the presence of a cytosine at alocation known to normally contain cytosine reflects that the originalcytosine was methylated.

Following bisulfite conversion, and PCR amplification, sequencescontaining a large number of unmethylated cytosines will have a lowcomplexity, since the non-methylated cytosines will have been convertedto thymine, and the resulting sequence will be dominated by only threebases (A, G, and T). Such low complexity sequences can be difficult tomap to a region (locus) of the genome. That is, when a low complexitynucleic acid is sequenced, it can be difficult to know what part of thegenome the sequence comes from. Such a problem is particularly acute invarious sequencing approaches that employ short read-lengths.

Bisulfite treatment is also problematic because of limited sample size.Treatment is harsh, and small amounts of starting material are noteasily analyzed using bisulfite.

SUMMARY

A method of quantitating methylation in a target nucleic acidcomprising;

treating a target nucleic acid with a MeDNA binding protein, wherein theMeDNA binding protein forms a blocking complex with a methylatedcytosine in the target nucleic acid, wherein the methylated cytosine inthe target nucleic acid is near a first target specific primer bindingsite;

extending a first target specific primer hybridized to the first targetspecific primer binding site to form a target nucleic acid extensionproduct;

degrading the target nucleic acid;

amplifying the target nucleic acid extension product;

determining the difference between the amount of the target nucleic acidwith the amount of a control nucleic acid lacking a methylated cytosine;and, quantitating methylation in the target nucleic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows one illustrative embodiment according to the presentteachings.

FIG. 2 shows one illustrative embodiment according to the presentteachings.

FIG. 3 shows one illustrative embodiment according to the presentteachings.

FIG. 4 shows one illustrative embodiment according to the presentteachings.

FIG. 5 shows one illustrative embodiment according to the presentteachings.

FIG. 6 shows one illustrative embodiment according to the presentteachings.

FIG. 7 shows one example according to the present teachings.

FIG. 8 shows effect of antibody by amount on Ct and delta Ct.

FIG. 9 shows effect of antibody amount/duplex reaction

FIG. 10 shows effect of antibody amount/duplex reaction: reduced Ctshift of 0 Me by increasing RT amount

FIG. 11 shows effect of antibody amount/duplex reaction with normalizedtemplate/high salt

DESCRIPTION OF EXEMPLARY EMBODIMENTS

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not intended to limit the scope of the current teachings. Inthis application, the use of the singular includes the plural unlessspecifically stated otherwise. Also, the use of “comprise”, “contain”,and “include”, or modifications of those root words, for example but notlimited to, “comprises”, “contained”, and “including”, are not intendedto be limiting. The use of “or” means “and/or” unless stated otherwise.The term and/or means that the terms before and after can be takentogether or separately. For illustration purposes, but not as alimitation, “X and/or Y” can mean “X” or “Y” or “X and Y”.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the described subject matter inany way. All literature and similar materials cited in this application,including, patents, patent applications, articles, books, treatises, andinternet web pages are expressly incorporated by reference in theirentirety for any purpose. In the event that one or more of theincorporated literature and similar defines or uses a term in such a waythat it contradicts that term's definition in this application, thisapplication controls. While the present teachings are described inconjunction with various embodiments, it is not intended that thepresent teachings be limited to such embodiments. On the contrary, thepresent teachings encompass various alternatives, modifications, andequivalents, as will be appreciated by those of skill in the art.

SOME DEFINITIONS

As used herein, the term “degrading” refers to removal of unwantednucleic acids in a reaction. Such degradation can be achieved, forexample, by employing first primers in the primer extension reactionthat contain a nuclease resistant blocking moiety, thus protectingextension products from nuclease degradation. Examples of suitableblocking moieties and nuclease-mediated approaches are known in the art,and are described for example in Chen et al., U.S. Pat. No. 7,208,278,Greenfield et al., U.S. patent application Ser. No. 10/202,211, andBarany et al., U.S. Pat. No. 6,797,470.

As used herein, the term “amplifying” refers to any process thatincreases the amount of a desired nucleic acid. Any of a variety ofknown amplification procedures can be employed in the present teachings,including PCR (see for example U.S. Pat. No. 4,683,202), as well as anyof a variety of ligation-mediated approaches, including LDR and LCR (seefor example U.S. Pat. No. 5,494,810, U.S. Pat. No. 5,830,711, U.S. Pat.No. 6,054,564). Some other amplification procedures include isothermalapproaches such as rolling circle amplification and helicase-dependantamplification.

As used herein, the term “blocking complex” refers to a structure formedby the interaction of a methylated cytosine with a MeDNA bindingprotein, as well as the structure formed by the interaction of anunmethylated cytosine with a non-MeDNA binding protein. Both situationsbring about the inability of a primer extension reaction to proceedgiven the presence of the blocking complex.

As used herein, the term “cytosine position of interest” refers to acytosine residue in a nucleic acid whose methylation status is relevantto the experimentalist.

As used herein, the term “near a primer binding site” and various usagesof it, refers to the location of a cytosine of interest in a nucleicacid, in reference to the position of a first primer. Thus, a cytosineof interest can be in the sequence of the primer binding site, or can be1, 2 or fewer, 3 or fewer, 4 or fewer, 5 or fewer, 6 or fewer, 7 orfewer, 8 or fewer, 9 or fewer, 10 or fewer, 11 or fewer, 12 or fewer, 13or fewer, 14 or fewer, 15 or fewer, 16 or fewer, 17 or fewer, 18 orfewer, 19 or fewer, 20 or fewer, 21 or fewer, 22 or fewer, 23 or fewer,24 or fewer, 25 or fewer, 26 or fewer, 27 or fewer, 28 or fewer, 29 orfewer, 30 or fewer, 30-40, 40-50, 50-60, 60-70, 70-100, 100-150,150-300, 300-500, or 500-1000, nucleotides away from the 3′ end of thefirst primer. The various embodiments depicted in FIGS. 3 and 6illustrate situations where the cytosine position of interest is near aprimer binding site.

As used herein, the term “cofactor” refers to compounds that binds to anenzyme to facilitate enzyme catalysis. In those embodiments employing aMeDNA binding protein, cofactors serve the function of modulate thebinding of the protein to DNA. In those embodiments employing anon-MeDNA binding protein, cofactors serve the function of providingtransferable chemical motif for the enzyme reaction.

As used herein, the term “first primer” refers generally to the primeremployed in the extension reaction. A first control specific primer isone example of a first primer. A first control specific primerhybridizes to a “first control specific primer site.” A first targetspecific primer is another example of a first primer. A first targetspecific primer hybridizes to a “first target specific primer site”.

As used herein, the term “second primer” refers generally to a primeremployed in a PCR, which hybridizes to the extension product produced inthe extension reaction, and which can extend to form a complementarystrand. In those embodiments in which the amplifying is a PCR, a firstprimer can hybridize to the complementary strand generated by extensionof the second primer, and itself become extended to effectuate the PCRprocess. A second control specific primer is one example of a secondprimer. A second control primer can hybridize to a “control specificextension product”. A second target specific primer is another exampleof a second primer. A second target specific primer hybridizes to a“target specific extension product”.

As used herein, the term “extension product” refers generally to theresult of a primer extension reaction. A target specific extensionproduct is one example of an extension product, it resulting fromextension of a first target specific primer. A control specificextension product is another example of an extension product, itresulting from extension of a first control specific primer.

As used herein, the term “MeDNA binding protein” refers to a proteinthat binds methylated cytosine in a nucleic acid, thus forming a bindingcomplex. Examples of MeDNA binding proteins include MeCP2, MBD1, MBD2,MBD3 and MBD4 (Fraga, M F, et al Nucleic Acid Research, 2003, 31(6),1765-1774), Dnmt1 (Cheng, X. and Blumenthal R M Structure, 2008, 16,341-350), methyl-CpG antibodies, recombinant proteins with multiplemethyl-DNA-binding domains (Jorgensen, H F. Et al Nucleic Acid Research,2006, 34(13), e96), McrBC, and VIM1 (Woo, H R et al Genes & Development,2007, 21, 267-277).

As used herein, the term “non MeDNA binding protein” refers to a proteinthat binds unmethylated cytosine in a nucleic acid, thus forming abinding complex. Examples of non-MeDNA biding proteins include Dnmt3aand Dnmt3b ((Cheng, X. and Blumenthal R M Structure, 2008, 16, 341-350),M. Sssl (Flynn, J. et al Biochemistry, 1996, 35, 7308-7315), CXXC domainof MDB1 (Voo, K S et al Mol. Cell Biol. 2000, 20, 2108-2021).

As used herein, the term “Ct value” refers to a cycle in a PCR at whicha particular intensity of a probe is observed. Examples of PCR analysisusing Ct values can be found in U.S. Pat. No. 7,132,239, U.S. Pat. No.7,057,025, U.S. Pat. No. 6,890,718, U.S. Pat. No. 5,952,202, U.S. Pat.No. 6,884,583, and U.S. Pat. No. 6,432,642.

MeDNA binding Protein Embodiments

In some embodiments, the present teachings provide a method ofquantitating methylation in a target nucleic acid. For example, in someembodiments, the present teachings provide a method of quantitatingmethylation in a target nucleic acid comprising; treating a targetnucleic acid with a MeDNA binding protein, wherein the MeDNA bindingprotein forms a blocking complex with a methylated cytosine in thetarget nucleic acid, wherein the methylated cytosine in the targetnucleic acid is near a first target specific primer binding site;extending a first target specific primer hybridized to the first targetspecific primer binding site to form a target nucleic acid extensionproduct; degrading the target nucleic acid; amplifying the targetnucleic acid extension product; determining the difference between theamount of the target nucleic acid with the amount of a control nucleicacid lacking a methylated cytosine; and, quantitating methylation in thetarget nucleic acid.

In some embodiments, the amplifying comprises a polymerase chainreaction comprising a first target specific primer and a second targetspecific primer.

In some embodiments, the degrading comprises treating the target nucleicacid with a nuclease, wherein the target nucleic acid extension productis resistant to the nuclease due to a blocking moiety in the firsttarget specific primer.

One illustrative embodiment is depicted in FIG. 1. Here, a targetnucleic acid (1) containing a methylated cytosine residue (M) is presentin a reaction mixture. A control nucleic acid (2) contains anunmethylated cytosine (C). The target nucleic acid can be treated with aMeDNA binding protein (oval shape), which can form a blocking complexwith a methylated cytosine in the target nucleic acid (3). The controlnucleic acid, lacking a methylated cytosine, fails to form a blockingcomplex with the MeDNA binding protein (note that the oval, representingthe MeDNA binding protein, is not bound with the unmethylated cytosine(C) in the control nucleic acid (2). A primer extension reaction can beattempted by hybridizing a first target specific primer (30) to thetarget nucleic acid, and a first control specific primer (31) to thecontrol nucleic acid. The target nucleic acid with the methylatedcytosine, due to the blocking complex with the MeDNA binding protein, isunable to undergo primer extension (4), producing for example truncatedextension products (6). However, the control nucleic acid with theunmethylated cytosine is able to undergo primer extension (5) due to theabsence of a blocking complex, thus forming a control nucleic acidextension product (dashed, (7)). The target nucleic acid and controlnucleic can then be degraded (note the absence of (1) and (2)), leavingextension products in tact. Amplifying the extension products can thenbe performed, here shown by a PCR using a first target specific primer(8) and a second target specific primer (9) for the target nucleic acid,and a first control specific primer (10) and a second control specificprimer (11) for the control nucleic acid. Determining the differencebetween the amount of the target nucleic acid with the amount of acontrol nucleic acid lacking a methylated cytosine allows forquantitating methylation in the target nucleic acid. Here, amplificationcan proceed on the control extension product, but fails on the truncatedtarget extension product.

Illustrative graphs arising from practice of the method of FIG. 1, aredepicted in FIG. 2. FIG. 2A shows the hypothetical results of anexperiment in which the target nucleic acid is more methylated than thecontrol nucleic acid. Here, the results of a real-time PCR illustratethat the Ct value for the control nucleic acid (12) is lower(left-shifted) than the Ct value for the target nucleic acid (13). Thisshift reflects the greater number of extension products resulting fromthe extension reaction of the control nucleic acid as compared to thetarget nucleic acid. This can be interpreted to reflect that the MeDNAbinding protein preferentially bound target nucleic acids containingmethylated cytosine to form blocking complexes, thus preventing theformation of target nucleic acid extension products. Thus, the targetnucleic acids contain a greater amount of methylated cytosine at thecytosine position of interest than do the control nucleic acids.

FIG. 2B shows the hypothetical results of an experiment in which thetarget nucleic acid is less methylated than the control nucleic acid.Here, the results of a real-time PCR illustrate that the Ct value forthe target nucleic acid (14) is lower (left-shifted) than the Ct valuefor the control nucleic acid (15). This shift reflects the greaternumber of extension products resulting from the extension reaction ofthe target nucleic acid as compared to the control nucleic acid. Thiscan be interpreted to reflect that the MeDNA binding proteinpreferentially bound control nucleic acids containing methylatedcytosine to form blocking complexes, thus preventing the formation ofcontrol nucleic acid extension products. Thus, the target nucleic acidscontain a lesser amount of methylated cytosine at the cytosine positionof interest than do the control nucleic acids.

FIG. 3 illustrates some various possible relationships between thelocation of a cytosine of interest, the blocking complex formed with theMeDNA binding protein, and the first primer. In some embodiments, thefirst primer hybridizes upstream from the blocking complex formed by theMeDNA binding protein and the methylated cytosine in the target nucleicacid, and further extension of the first primer is blocked. This isdepicted in FIG. 3A. Note that in FIG. 3A the first primer is shownhybridized (see vertical lines indicating Hydrogen bonds) upstream fromthe cytosine of interest, and that the primer is partially extended(dashed horizontal line). However, the blocking complex between themethylated cytosine and the MeDNA binding protein blocks furtherextension of the first primer. In some embodiments, the first primerhybridizes on the methylated cytosine on the blocking complex formed bythe MeDNA binding protein and the methylated cytosine in the targetnucleic acid, and initial extension of the first primer is blocked. Thisis depicted in FIG. 3B. Note than in FIG. 3B the first primer is shownhybridized (see the vertical lines indicating Hydrogen bonds) on thecytosine of interest, and that the primer is not extended. The blockingcomplex between the methylated cytosine and the MeDNA binding proteinblocks initial extension of the first primer. In some embodiments, thefirst primer does not hybridize to the methylated cytosine on theblocking complex formed by the MeDNA binding protein and the methylatedcytosine in the target nucleic acid. This is depicted in FIG. 3C. Notethe absence of vertical lines between the first primer and the targetnucleic acid, indicating that the blocking complex formed by the MeDNAbinding protein and the methylated cytosine in the target nucleic acidprevents the first primer from hybridizing. As will be appreciated byone of ordinary skill in the art in light of the present teachings, thechoice of position between the cytosine of interest, the MeDNA bindingprotein, and the primer binding site can be chosen according to theexperimentalist using routine experimentation.

The control nucleic acid can be employed in a variety of ways. Forexample, the control nucleic acid can be in the same reaction mixture asthe target nucleic acid and can be a different sequence than the targetnucleic acid. The control nucleic acid can be of a known concentration,and can be known to contain an unmethylated cytosine or a particularamount of unmethylated cytosine at the position of interest. In someembodiments, the control nucleic acid can be in a different reactionmixture from the target nucleic acid. For example, the control nucleicacid can be the same sequence as the target nucleic acid, and can bepresent in a known amount in the different reaction mixture. In someembodiments, the control nucleic acid can be a different sequence thanthe target nucleic acid, and can be present in a known amount in thedifferent reaction mixture. Various methods of performing the controlreactions will be appreciated by one of skill in the art in light of thepresent teachings, including for example employing controls of theappropriate abundance class (see Bodeau et al., U.S. patent applicationSer. No. 11/372,242.

The present teachings also provide a method of quantitating methylationin a target nucleic acid comprising;

treating, in any order,

-   -   (a) the target nucleic acid with a MeDNA binding protein,        wherein the MeDNA binding protein forms a blocking complex with        a methylated cytosine in the target nucleic acid, wherein the        methylated cytosine is disposed between a first target specific        primer binding site and a second target specific primer binding        site; and,    -   (b) a control nucleic acid with a MeDNA binding protein, wherein        the MeDNA binding protein fails to form a blocking complex with        an unmethylated cytosine in the control nucleic acid, wherein        the non-methylated cytosine is disposed between a first control        specific primer binding site and a second control specific        primer binding site;    -   extending, in any order,    -   (a) a first target specific primer hybridized to the first        target specific primer binding site to form a target nucleic        acid extension product; and,    -   (b) a first control specific primer hybridized to the first        control specific primer binding site to form a control nucleic        acid extension product;    -   degrading, in any order,    -   (a) the target nucleic acid; and,    -   (b) the control nucleic acid;    -   amplifying, in any order,    -   (a) the target nucleic acid extension product in a polymerase        chain reaction comprising a first target specific primer and a        second target specific primer; and,    -   (b) the control nucleic acid extension product in a polymerase        chain reaction comprising a first control specific primer and a        second control specific primer;

determining the difference between the amount of target nucleic acidwith the amount of control nucleic acid; and,

quantitating methylation in the target nucleic acid.

In some embodiments, the determining comprises;

measuring, in any order,

-   -   (a) a first Ct value associated with the amount of the target        nucleic acid, and,    -   (b) a second Ct value associated with the amount of the control        nucleic acid; and,

quantitating methylation in the target nucleic by comparing the first Ctvalue with the second Ct value.

-   -   In some embodiments, the first Ct value is higher than the        second Ct value, and the target nucleic acid is more methylated        than the control nucleic acid.

In some embodiments, the first Ct value is lower than the second Ctvalue, and the target nucleic acid is less methylated than the controlnucleic acid.

In some embodiments, the target nucleic acid and the control nucleicacid comprise the same first primer binding site and the same secondprimer binding site.

In some embodiments, the target nucleic acid is amplified in a separatereaction vessel from the control nucleic acid.

In some embodiments, the target nucleic acid and the control nucleicacid comprise the same first primer binding site and the same secondprimer binding site and are amplified with a common first primer and acommon second primer.

In some embodiments, the target nucleic acid is amplified in a samereaction vessel as the control nucleic acid.

In some embodiments, the target nucleic acid and the control nucleicacid comprise a different first primer binding site and a differentsecond primer binding site and are amplified with a different firstprimer and a different second primer.

In some embodiments, the quantitating comprises measuring aninterchelating dye.

In some embodiments, the determining comprises;

measuring displacement of a target sequence specific probe, wherein thetarget sequence specific probe hybridizes to a region of the targetnucleic acid extension product, or complement to the target nucleic acidextension product, disposed between the first target specific primerbinding site and the second target specific primer binding site;

measuring displacement of a control sequence specific probe, wherein thecontrol sequence specific probe hybridizes to a region of the controlnucleic acid extension product, or complement to the control nucleicacid extension product, disposed between the first control specificprimer binding site and the second control specific primer binding site.

In some embodiments, the treating with the MeDNA binding protein furthercomprises a cofactor.

In some embodiments, the cofactor is SELECTED FROM THE GROUP CONSISTINGOF S-adenosylmethionine, S-adenosylhomocysteine and sinefungin. In apreferred embodiment, S-adenosylhomocycteine is used.

In some embodiments, the first primer hybridizes upstream from theblocking complex formed by the MeDNA binding protein and the methylatedcytosine in the target nucleic acid, and further extension of the firstprimer is blocked.

In some embodiments, the first primer hybridizes on the methylatedcytosine on the blocking complex formed by the MeDNA binding protein andthe methylated cytosine in the target nucleic acid, and initialextension of the first primer is blocked.

In some embodiments, the first primer does not hybridize to themethylated cytosine on the blocking complex formed by the MeDNA bindingprotein and the methylated cytosine in the target nucleic acid.

Non-MeDNA Binding Protein Embodiments

In some embodiments, the present teachings provide a method ofquantitating methylation in a target nucleic acid. For example, in someembodiments, the present teachings provide a method of quantitatingmethylation in a target nucleic acid comprising; treating a controlnucleic acid with a non-MeDNA binding protein, wherein the non-MeDNAbinding protein forms a blocking complex with an unmethylated cytosinein the control nucleic acid, wherein the unmethylated cytosine in thecontrol nucleic acid is near a first control specific primer bindingsite; extending a first primer hybridized to the first primer bindingsite to form a control nucleic acid extension product; degrading thecontrol nucleic acid; amplifying the control nucleic acid extensionproduct; determining the difference between the amount of the controlnucleic acid with the amount of a target nucleic acid containing amethylated cytosine; and, quantitating methylation in the target nucleicacid.

In some embodiments, the amplifying comprises a polymerase chainreaction comprising a first target specific primer and a second targetspecific primer.

In some embodiments, the degrading comprises treating the target nucleicacid with a nuclease, wherein the target nucleic acid extension productis resistant to the nuclease due to a blocking moiety in the firsttarget specific primer.

One illustrative embodiment is depicted in FIG. 4. Here, a targetnucleic acid (16) containing a methylated cytosine residue (M) ispresent in a reaction mixture. A control nucleic acid (17) contains anunmethylated cytosine (C). The target nucleic acid can be treated with anon-MeDNA binding protein (oval shape), but due to the presence of amethyl group on the cytosine of interest, fails to form a blockingcomplex with the non-MeDNA binding protein (note that the oval shape,representing the non-MeDNA binding protein, is not bound with themethylated cytosine (C) in the target nucleic acid (16)). The controlnucleic acid, lacking a methylated cytosine, is able to form a blockingcomplex (18) with the non-MeDNA binding protein (note that the oval,representing the MeDNA binding protein, is bound with the unmethylatedcytosine (C) in the control nucleic acid (17). A primer extensionreaction can be performed. The control nucleic acid with theunmethylated cytosine, due to the blocking complex with the non-MeDNAbinding protein, is unable to undergo primer extension (19) with a firstprimer (33), producing for example truncated extension products (20).However, the target nucleic acid with the methylated cytosine is able toundergo primer extension with a first primer (32), thus forming a targetnucleic acid extension product (dashed, (21)). The target nucleic acidand control nucleic can then be degraded (note the absence of (16) and(17), leaving behind any extension products. Amplifying the extensionproducts can then be performed, here shown as a PCR using a first targetspecific primer (22) and a second target specific primer (23) for thetarget nucleic acid, and a first control specific primer (24) and asecond control specific primer (25) for the control nucleic acid.Determining the difference between the amount of the target nucleic acidwith the amount of a control nucleic acid lacking a methylated cytosineallows for quantitating methylation in the target nucleic acid. Here,amplification can proceed on the target extension product, but fails onthe truncated control extension product.

Representative graphs arising from practicing the method of FIG. 4, aredepicted in FIG. 5. FIG. 5A shows the results of an experiment in whichthe target nucleic acid is more methylated than the control nucleicacid. Here, the results of a real-time PCR illustrate that the Ct valuefor the control nucleic acid (26) is higher (right-shifted) than the Ctvalue for the target nucleic acid (27). This shift reflects the greaternumber of extension products resulting from the extension reaction ofthe methylated target nucleic acid as compared to the control nucleicacid. This can be interpreted to reflect that the non-MeDNA bindingprotein preferentially bound control nucleic acids containingunmethylated cytosine, thus preventing the formation of control nucleicacid extension products. Thus, the target nucleic acids contain agreater amount of methylated cytosine at the cytosine position ofinterest than do the control nucleic acids.

FIG. 5B shows the results of an experiment in which the target nucleicacid is less methylated than the control nucleic acid. Here, the resultsof a real-time PCR illustrate that the Ct value for the control nucleicacid (28) is lower (left-shifted) than the Ct value for the targetnucleic acid (29). This shift reflects the greater number of extensionproducts resulting from the extension reaction of the control nucleicacid as compared to the target nucleic acid. This can be interpreted toreflect that the non-MeDNA binding protein preferentially bound targetnucleic acids containing unmethylated cytosine, thus preventing theformation of target nucleic acid extension products. Thus, the targetnucleic acids contain a lesser amount of methylated cytosine at thecytosine position of interest than do the control nucleic acids.

FIG. 6 illustrates the various possible relationships between thelocation of a cytosine of interest, the blocking complex formed with theMeDNA binding protein, and the first primer. In some embodiments, thefirst primer hybridizes upstream from the blocking complex formed by thenon-MeDNA binding protein and the unmethylated cytosine in the targetnucleic acid, and further extension of the first primer is blocked. Thisis depicted in FIG. 6A. Note that in FIG. 6A the first primer is shownhybridized (see vertical lines indicating Hydrogen bonds) upstream fromthe cytosine of interest, and that the primer is partially extended(dashed horizontal line). However, the blocking complex between theunmethylated cytosine and the non-MeDNA binding protein blocks furtherextension of the first primer. In some embodiments, the first primerhybridizes on the unmethylated cytosine on the blocking complex formedby the non-MeDNA binding protein and the unmethylated cytosine in thetarget nucleic acid, and initial extension of the first primer isblocked. This is depicted in FIG. 6B. Note than in FIG. 6B the firstprimer is shown hybridized (see the vertical lines indicating Hydrogenbonds) on the cytosine of interest, and that the primer is not extended.The blocking complex between the unmethylated cytosine and the non-MeDNAbinding protein blocks initial extension of the first primer. In someembodiments, the first primer does not hybridize to the unmethylatedcytosine on the blocking complex formed by the non-MeDNA binding proteinand the unmethylated cytosine in the target nucleic acid. This isdepicted in FIG. 6C. Note the absence of vertical lines between thefirst primer and the target nucleic acid, indicating that the blockingcomplex formed by the non-MeDNA binding protein and the unmethylatedcytosine in the target nucleic acid prevents the first primer fromhybridizing. As will be appreciated by one of ordinary skill in the artin light of the present teachings, the choice of position between thecytosine of interest, the non-MeDNA binding protein, and the primerbinding site can be chosen according to the experimentalist usingroutine experimentation.

In view of FIG. 4, the control nucleic acid can be employed in a varietyof ways. For example, the control nucleic acid can be in the samereaction mixture as the target nucleic acid and can be a differentsequence than the target nucleic acid. The control nucleic acid can beof a known concentration, and can be known to contain an unmethylatedcytosine at the position of interest. In some embodiments, the controlnucleic acid can be in a different reaction mixture from the targetnucleic acid. For example, the control nucleic acid can be the samesequence as the target nucleic acid, and can be present in a knownamount in the different reaction mixture. In some embodiments, thecontrol nucleic acid can be a different sequence than the target nucleicacid, and can be present in a known amount in the different reactionmixture. Various methods of performing the control reactions will beappreciated by one of skill in the art in light of the presentteachings, including for example employing controls of the appropriateabundance class (see Bodeau et al., U.S. patent application Ser. No.11/372,242).

The present teachings also provide a method of quantitating methylationin a target nucleic acid comprising;

treating, in any order,

-   -   (a) the control nucleic acid with a non-MeDNA binding protein,        wherein the non-MeDNA binding protein forms a blocking complex        with an unmethylated cytosine in the control nucleic acid        lacking, wherein the unmethylated cytosine is disposed between a        first control-specific primer binding site and a second        control-specific primer binding site; and,    -   (b) a target nucleic acid with a non-MeDNA binding protein,        wherein the non-MeDNA binding protein fails to form a blocking        complex with a a methylated cytosine in the target nucleic acid,        wherein the methylated cytosine is disposed between a first        target specific primer binding site and a second target specific        primer binding site;

extending, in any order,

-   -   (a) a first target specific primer hybridized to the first        target specific primer binding site to form a target nucleic        acid extension product; and,    -   (b) a first control specific primer hybridized to the first        control specific primer binding site to form a control nucleic        acid extension product;

degrading, in any order,

-   -   (a) the target nucleic acid; and,    -   (b) the control nucleic acid;

amplifying, in any order,

-   -   (a) the target nucleic acid extension product in a polymerase        chain reaction comprising a first target specific primer and a        second target specific primer; and,    -   (b) the control nucleic acid extension product in a polymerase        chain reaction comprising a first control specific primer and a        second control specific primer;

determining the difference between the amount of target nucleic acidwith the amount of control nucleic acid; and,

quantitating methylation in the target nucleic acid.

In some embodiments, the determining comprises;

measuring, in any order,

-   -   (a) a first Ct value associated with the amount of the target        nucleic acid, and,    -   (b) a second Ct value associated with the amount of the control        nucleic acid; and,

quantitating methylation in the target nucleic by comparing the first Ctvalue with the second Ct value.

In some embodiments, the first Ct value is higher than the second Ctvalue, and the target nucleic acid is less methylated than the controlnucleic acid.

In some embodiments, the first Ct value is lower than the second Ctvalue, and the target nucleic acid is more methylated than the controlnucleic acid.

In some embodiments, the target nucleic acid and the control nucleicacid comprise the same first primer binding site and the same secondprimer binding site.

In some embodiments, the target nucleic acid is amplified in a separatereaction vessel from the control nucleic acid.

In some embodiments, the target nucleic acid and the control nucleicacid comprise the same first primer binding site and the same secondprimer binding site and are amplified with a common first primer and acommon second primer.

In some embodiments, the target nucleic acid is amplified in a samereaction vessel as the control nucleic acid.

In some embodiments, the target nucleic acid and the control nucleicacid comprise a different first primer binding site and a differentsecond primer binding site and are amplified with a different firstprimer and a different second primer.

In some embodiments, the quantitating comprises measuring aninterchelating dye.

In some embodiments, the determining comprises;

measuring displacement of a target sequence specific probe, wherein thetarget sequence specific probe hybridizes to a region of the targetnucleic acid extension product, or complement to the target nucleic acidextension product, disposed between the first target specific primerbinding site and the second target specific primer binding site;

measuring displacement of a control sequence specific probe, wherein thecontrol sequence specific probe hybridizes to a region of the controlnucleic acid extension product, or complement to the control nucleicacid extension product, disposed between the first control specificprimer binding site and the second control specific primer binding site.

In some embodiments, the treating with the non-MeDNA binding proteinfurther comprises a cofactor.

In some embodiments, the cofactor is selected from the group consistingof S-adenosylmethionine, S-adenosylhomocysteine and sinefungin. In apreferred embodiment, S-adenosylhomocycteine is used.

In some embodiments, the first primer hybridizes upstream from theblocking complex formed by the non-MeDNA binding protein and theunmethylated cytosine in the control nucleic acid, and further extensionof the first primer is blocked.

In some embodiments, the first primer hybridizes on the unmethylatedcytosine on the blocking complex formed by the non-MeDNA binding proteinand the unmethylated cytosine in the control nucleic acid, and initialextension of the first primer is blocked.

In some embodiments, the first primer does not hybridize to theunmethylated cytosine on the blocking complex formed by the non-MeDNAbinding protein and the unmethylated cytosine in the target nucleicacid.

Kits

The instant teachings also provide kits designed to expedite performingcertain of the disclosed methods. Kits may serve to expedite theperformance of certain disclosed methods by assembling two or morecomponents required for carrying out the methods. In certainembodiments, kits contain components in pre-measured unit amounts tominimize the need for measurements by end-users. In some embodiments,kits include instructions for performing one or more of the disclosedmethods. Preferably, the kit components are optimized to operate inconjunction with one another.

MeDNA Binding Protein Kits

In some embodiments, the present teachings provide a kit forquantitating methylation in a target nucleic acid comprising;

a Me-DNA binding protein;

a first target specific primer;

a second target specific primer;

a first control specific primer;

a second control specific primer; and,

a polymerase.

In some embodiments of the kit,

the first control specific primer is a different sequence from the firsttarget specific primer; and,

the second control specific primer is a different sequence from thesecond target specific primer.

In some embodiments of the kit,

the first control specific primer is a same sequence as the first targetspecific primer; and,

the second control specific primer is a same sequence as the secondtarget specific primer.

In some embodiments, the kit further comprises a control sequencespecific probe.

In some embodiments, the kit further comprises a target sequencespecific probe, wherein the control sequence specific probe is adifferent sequence from the target sequence specific probe.

In some embodiments, the kit further comprises a target sequencespecific probe, wherein the control sequence specific probe is a samesequence as the target sequence specific probe.

In some embodiments, the kit comprises a cofactor.

Non-MeDNA Binding Protein Kits

In some embodiments, the present teachings provide a kit forquantitating methylation in a target nucleic acid comprising;

a non-MeDNA binding protein;

a first target specific primer;

a second target specific primer;

a first control specific primer;

a second control specific primer; and,

a polymerase.

In some embodiments of the kit,

the first control specific primer is a different sequence from the firsttarget specific primer; and,

the second control specific primer is a different sequence from thesecond target specific primer.

In some embodiments, the first control specific primer is a samesequence as the first target specific primer; and,

-   -   the second control specific primer is a same sequence as the        second target specific primer.

In some embodiments, the kit comprises a control sequence specificprobe.

In some embodiments, the kit comprises a target sequence specific probe,wherein the control sequence specific probe is a different sequence fromthe target sequence specific probe.

In some embodiments, the kit comprises a target sequence specific probe,wherein the control sequence specific probe is a same sequence as thetarget sequence specific probe.

In some embodiments, the kit comprises a cofactor.

Although the disclosed teachings have been described with reference tovarious applications, methods, and kits, it will be appreciated thatvarious changes and modifications may be made without departing from theteachings herein. The foregoing examples are provided to betterillustrate the present teachings and are not intended to limit the scopeof the teachings herein. Certain aspects of the present teachings may befurther understood in light of the following claims.

1. A method of quantitating methylation in a target nucleic acidcomprising; treating a target nucleic acid with a MeDNA binding protein,wherein the MeDNA binding protein forms a blocking complex with amethylated cytosine in the target nucleic acid, wherein the methylatedcytosine in the target nucleic acid is near a first target specificprimer binding site; extending a first target specific primer hybridizedto the first target specific primer binding site to form a targetnucleic acid extension product; degrading the target nucleic acid;amplifying the target nucleic acid extension product; determining thedifference between the amount of the target nucleic acid with the amountof a control nucleic acid lacking a methylated cytosine; and,quantitating methylation in the target nucleic acid.
 2. The methodaccording to claim 1 wherein the amplifying comprises a polymerase chainreaction comprising a first target specific primer and a second targetspecific primer.
 3. The method according to claim 1 wherein thedegrading comprises treating the target nucleic acid with a nuclease,wherein the target nucleic acid extension product is resistant to thenuclease due to a blocking moiety in the first target specific primer.4. A method of quantitating methylation in a target nucleic acidcomprising; treating, in any order, (a) the target nucleic acid with aMeDNA binding protein, wherein the MeDNA binding protein forms ablocking complex with a methylated cytosine in the target nucleic acid,wherein the methylated cytosine is disposed between a first targetspecific primer binding site and a second target specific primer bindingsite; and, (b) a control nucleic acid with a MeDNA binding protein,wherein the MeDNA binding protein fails to form a blocking complex withan unmethylated cytosine in the control nucleic acid, wherein thenon-methylated cytosine is disposed between a first control specificprimer binding site and a second control specific primer binding site;extending, in any order, (a) a first target specific primer hybridizedto the first target specific primer binding site to form a targetnucleic acid extension product; and, (b) a first control specific primerhybridized to the first control specific primer binding site to form acontrol nucleic acid extension product; degrading, in any order, (a) thetarget nucleic acid; and, (b) the control nucleic acid; amplifying, inany order, (a) the target nucleic acid extension product in a polymerasechain reaction comprising a first target specific primer and a secondtarget specific primer; and, (b) the control nucleic acid extensionproduct in a polymerase chain reaction comprising a first controlspecific primer and a second control specific primer; determining thedifference between the amount of target nucleic acid with the amount ofcontrol nucleic acid; and, quantitating methylation in the targetnucleic acid.
 5. The method of claim 4 wherein the determiningcomprises; measuring, in any order, (a) a first Ct value associated withthe amount of the target nucleic acid, and, (b) a second Ct valueassociated with the amount of the control nucleic acid; and,quantitating methylation in the target nucleic by comparing the first Ctvalue with the second Ct value.
 6. The method of claim 4 wherein thefirst Ct value is higher than the second Ct value, and the targetnucleic acid is more methylated than the control nucleic acid.
 7. Themethod of claim 4 wherein the first Ct value is lower than the second Ctvalue, and the target nucleic acid is less methylated than the controlnucleic acid.
 8. The method of claim 4 wherein the target nucleic acidand the control nucleic acid comprise the same first primer binding siteand the same second primer binding site.
 9. The method according toclaim 4 wherein the target nucleic acid is amplified in a separatereaction vessel from the control nucleic acid.
 10. The method of claim 9wherein the target nucleic acid and the control nucleic acid comprisethe same first primer binding site and the same second primer bindingsite and are amplified with a common first primer and a common secondprimer.
 11. The method according to claim 4 wherein the target nucleicacid is amplified in a same reaction vessel as the control nucleic acid.12. The method of claim 11 wherein the target nucleic acid and thecontrol nucleic acid comprise a different first primer binding site anda different second primer binding site and are amplified with adifferent first primer and a different second primer.
 13. The methodaccording to claim 4 wherein the quantitating comprises measuring aninterchelating dye.
 14. The method according to claim 4 wherein thedetermining comprises; measuring displacement of a target sequencespecific probe, wherein the target sequence specific probe hybridizes toa region of the target nucleic acid extension product, or complement tothe target nucleic acid extension product, disposed between the firsttarget specific primer binding site and the second target specificprimer binding site; measuring displacement of a control sequencespecific probe, wherein the control sequence specific probe hybridizesto a region of the control nucleic acid extension product, or complementto the control nucleic acid extension product, disposed between thefirst control specific primer binding site and the second controlspecific primer binding site.
 15. The method according to claim 4,wherein the treating with the MeDNA binding protein further comprises acofactor.
 16. The method according to claim 15 wherein the cofactor isselected from the group consisting of S-adenosylmethionine,S-adenosylhomocysteine and sinefungin. In a preferred embodiment,S-adenosylhomocycteine is used.
 17. The method according to claim 4wherein the first primer hybridizes upstream from the blocking complexformed by the MeDNA binding protein and the methylated cytosine in thetarget nucleic acid, and further extension of the first primer isblocked.
 18. The method according to claim 4 wherein the first primerhybridizes on the methylated cytosine on the blocking complex formed bythe MeDNA binding protein and the methylated cytosine in the targetnucleic acid, and initial extension of the first primer is blocked. 19.The method according to claim 4 wherein the first primer does nothybridize to the methylated cytosine on the blocking complex formed bythe MeDNA binding protein and the methylated cytosine in the targetnucleic acid.
 20. A kit for quantitating methylation in a target nucleicacid comprising; a Me-DNA binding protein; a first target specificprimer; a second target specific primer; a first control specificprimer; a second control specific primer; and, a polymerase.
 21. The kitaccording to claim 20 wherein; the first control specific primer is adifferent sequence from the first target specific primer; and, thesecond control specific primer is a different sequence from the secondtarget specific primer.
 22. The kit according to claim 20 wherein; thefirst control specific primer is a same sequence as the first targetspecific primer; and, the second control specific primer is a samesequence as the second target specific primer.
 23. The kit according toclaim 20 further comprising a control sequence specific probe.
 24. Thekit according to claim 23 further comprising a target sequence specificprobe, wherein the control sequence specific probe is a differentsequence from the target sequence specific probe.
 25. The kit accordingto claim 23 further comprising a target sequence specific probe, whereinthe control sequence specific probe is a same sequence as the targetsequence specific probe.
 26. The kit according to claim 20 furthercomprising a cofactor.
 27. The kit according to claim 26 wherein thecofactor is selected from the group consisting of S-adenosylmethionine,S-adenosylhomocysteine and sinefungin.
 28. A method of quantitatingmethylation in a target nucleic acid comprising; treating a controlnucleic acid with a non-MeDNA binding protein, wherein the non-MeDNAbinding protein forms a blocking complex with an unmethylated cytosinein the control nucleic acid, wherein the unmethylated cytosine in thecontrol nucleic acid is near a first control specific primer bindingsite; extending a first primer hybridized to the first primer bindingsite to form a control nucleic acid extension product; degrading thecontrol nucleic acid; amplifying the control nucleic acid extensionproduct in a polymerase chain reaction comprising a first controlspecific primer and a second control specific primer; determining thedifference between the amount of the control nucleic acid with theamount of a target nucleic acid containing a methylated cytosine; and,quantitating methylation in the target nucleic acid.
 29. The methodaccording to claim 28 wherein the amplifying comprises a polymerasechain reaction comprising a first target specific primer and a secondtarget specific primer.
 30. The method according to claim 28 wherein thedegrading comprises treating the target nucleic acid with a nuclease,wherein the target nucleic acid extension product is resistant to thenuclease due to a blocking moiety in the first target specific primer.31. A method of quantitating methylation in a target nucleic acidcomprising; treating, in any order, (a) the control nucleic acid with anon-MeDNA binding protein, wherein the non-MeDNA binding protein forms ablocking complex with an unmethylated cytosine in the control nucleicacid lacking, wherein the unmethylated cytosine is disposed between afirst control-specific primer binding site and a second control-specificprimer binding site; and, (b) a target nucleic acid with a non-MeDNAbinding protein, wherein the non-MeDNA binding protein fails to form ablocking complex with a a methylated cytosine in the target nucleicacid, wherein the methylated cytosine is disposed between a first targetspecific primer binding site and a second target specific primer bindingsite; extending, in any order, (a) a first target specific primerhybridized to the first target specific primer binding site to form atarget nucleic acid extension product; and, (b) a first control specificprimer hybridized to the first control specific primer binding site toform a control nucleic acid extension product; degrading, in any order,(a) the target nucleic acid; and, (b) the control nucleic acid;amplifying, in any order, (a) the target nucleic acid extension productin a polymerase chain reaction comprising a first target specific primerand a second target specific primer; and, (b) the control nucleic acidextension product in a polymerase chain reaction comprising a firstcontrol specific primer and a second control specific primer;determining the difference between the amount of target nucleic acidwith the amount of control nucleic acid; and, quantitating methylationin the target nucleic acid.
 32. The method of claim 31 wherein thedetermining comprises; measuring, in any order, (a) a first Ct valueassociated with the amount of the target nucleic acid, and, (b) a secondCt value associated with the amount of the control nucleic acid; and,quantitating methylation in the target nucleic by comparing the first Ctvalue with the second Ct value.
 33. The method of claim 31 wherein thefirst Ct value is higher than the second Ct value, and the targetnucleic acid is less methylated than the control nucleic acid.
 34. Themethod of claim 31 wherein the first Ct value is lower than the secondCt value, and the target nucleic acid is more methylated than thecontrol nucleic acid.
 35. The method of claim 31 wherein the targetnucleic acid and the control nucleic acid comprise the same first primerbinding site and the same second primer binding site.
 36. The methodaccording to claim 31 wherein the target nucleic acid is amplified in aseparate reaction vessel from the control nucleic acid.
 37. The methodof claim 36 wherein the target nucleic acid and the control nucleic acidcomprise the same first primer binding site and the same second primerbinding site and are amplified with a common first primer and a commonsecond primer.
 38. The method according to claim 31 wherein the targetnucleic acid is amplified in a same reaction vessel as the controlnucleic acid.
 39. The method of claim 38 wherein the target nucleic acidand the control nucleic acid comprise a different first primer bindingsite and a different second primer binding site and are amplified with adifferent first primer and a different second primer.
 40. The methodaccording to claim 31 wherein the quantitating comprises measuring aninterchelating dye.
 41. The method according to claim 31 wherein thedetermining comprises; measuring displacement of a target sequencespecific probe, wherein the target sequence specific probe hybridizes toa region of the target nucleic acid extension product, or complement tothe target nucleic acid extension product, disposed between the firsttarget specific primer binding site and the second target specificprimer binding site; measuring displacement of a control sequencespecific probe, wherein the control sequence specific probe hybridizesto a region of the control nucleic acid extension product, or complementto the control nucleic acid extension product, disposed between thefirst control specific primer binding site and the second controlspecific primer binding site.
 42. The method according to claim 31,wherein the treating with the non-MeDNA binding protein furthercomprises a cofactor.
 43. The method according to claim 42 wherein thecofactor is SELECTED FROM THE GROUP CONSISTING OF S-adenosylmethionine,S-adenosylhomocysteine and sinefungin. In a preferred embodiment,S-adenosylhomocycteine is used.
 44. The method according to claim 31wherein the first primer hybridizes upstream from the blocking complexformed by the non-MeDNA binding protein and the unmethylated cytosine inthe control nucleic acid, and further extension of the first primer isblocked.
 45. The method according to claim 31 wherein the first primerhybridizes on the unmethylated cytosine on the blocking complex formedby the non-MeDNA binding protein and the unmethylated cytosine in thecontrol nucleic acid, and initial extension of the first primer isblocked.
 46. The method according to claim 31 wherein the first primerdoes not hybridize to the unmethylated cytosine on the blocking complexformed by the non-MeDNA binding protein and the unmethylated cytosine inthe target nucleic acid.
 47. A kit for quantitating methylation in atarget nucleic acid comprising; a non-MeDNA binding protein; a firsttarget specific primer; a second target specific primer; a first controlspecific primer; a second control specific primer; and, a polymerase.48. The kit according to claim 47 wherein; the first control specificprimer is a different sequence from the first target specific primer;and, the second control specific primer is a different sequence from thesecond target specific primer.
 49. The kit according to claim 47wherein; the first control specific primer is a same sequence as thefirst target specific primer; and, the second control specific primer isa same sequence as the second target specific primer.
 50. The kitaccording to claim 47 further comprising a control sequence specificprobe.
 51. The kit according to claim 50 further comprising a targetsequence specific probe, wherein the control sequence specific probe isa different sequence from the target sequence specific probe.
 52. Thekit according to claim 50 further comprising a target sequence specificprobe, wherein the control sequence specific probe is a same sequence asthe target sequence specific probe.
 53. The kit according to claim 47further comprising a cofactor.
 54. The kit according to claim 53 whereinthe cofactor is selected from the group consisting ofS-adenosylmethionine, S-adenosylhomocysteine and sinefungin.